Assay devices and method of detecting a target analyte

ABSTRACT

Assay devices are provided including a receptacle having a sample entry port; a plunger disposed within the receptacle; at least one reagent; a membrane attached to a first surface of the plunger; a wick providing capillary force; and a detection zone. The membrane includes a target conjugate zone in which affinity components are disposed. Capture compounds are immobilized in a detection zone, which is located between the target conjugate zone and the wick. A method of detecting a target analyte is also provided, including providing the assay device; providing a sample suspected to contain a target analyte; adding the sample onto the device; allowing the sample to travel along the membrane until the sample reaches the detection zone; immobilizing the target analyte through reaction of the target analyte with the capture compounds; reacting the immobilized target analyte with a reagent to generate a detectable signal; and detecting the generated signal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofPCT/US2015/034624, filed Jun. 8, 2015, which claims the benefit of U.S.Application No. 62/009,602, filed Jun. 9, 2014, the disclosure of whichis incorporated by reference in its/their entirety herein.

FIELD

Assay devices and methods of detecting target analytes are providedincluding quantitative test results, such as using chemiluminescence,fluorescence, colorimetry, etc.

BACKGROUND

Lateral flow immunoassays are widely used in the food and medicaldiagnostics industries, as well as in simple point-of-care tests such asrapid strep tests and pregnancy tests. The lateral flow strip itself isusually a surfactant-infused nitrocellulose membrane, which contributesvariability to the device results. Current technology for immunoassaysis typically non-quantitative and lacks sensitivity.

Hence, there remains a need for an assay device that provides a rapidtest result while also providing quantitative results.

SUMMARY

Assay devices and methods of detecting a target analyte are provided. Ina first aspect, an assay device is provided. More particularly, an assaydevice is provided including a receptacle including a sample entry port;a plunger disposed within the receptacle and having a first surface; atleast one reagent; a membrane attached to the first surface of theplunger; a wick providing capillary force; and a detection zone. Themembrane includes a target conjugate zone in which a plurality ofaffinity components are disposed. A plurality of capture compounds areimmobilized in a detection zone, which is located between the targetconjugate zone and the wick.

In a second aspect, another assay device is provided. The assay deviceincludes a receptacle including a surface and a sample entry port; amembrane attached to the surface of the receptacle; at least one reagentdisposed in the receptacle; a plunger disposed within the receptacle andhaving a surface; and a detection zone in which a plurality of capturecompounds are immobilized. The membrane has a wick providing capillaryforce. The detection zone is attached to the surface of the plunger andis located between the sample entry port and the wick.

In a third aspect, a method of detecting a target analyte is provided.The method includes (a) providing an assay device including a receptacleincluding a surface and a sample entry port; at least one reagent; aplunger disposed within the receptacle and having a surface; a membraneattached to the surface of the plunger, the surface of the receptacle,or both; and a detection zone in which a plurality of capture compoundsare immobilized. The membrane includes a wick providing capillary force,and the detection zone is located between the sample entry port and thewick. The method further includes (b) providing a sample suspected tocontain a target analyte; (c) adding the sample onto the membranethrough the sample entry port; (d) allowing the sample to travel alongthe membrane until the sample reaches the detection zone; (e)immobilizing the target analyte through reaction of the target analytewith the capture compounds; (f) reacting the immobilized target analytewith the at least one reagent to generate a detectable signal; and (g)detecting the generated signal.

Use of the assay devices and methods allows for simple, rapid,quantitative assay testing of target analytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an exemplary partial cross-sectional schematic of an assaydevice.

FIG. 1B is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 1A following depression of the plunger.

FIG. 2A is another exemplary partial cross-sectional schematic of anassay device.

FIG. 2B is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 2A following rotation of the plunger.

FIG. 2C is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 2B following depression of the plunger.

FIG. 3A is a further exemplary partial cross-sectional schematic of anassay device.

FIG. 3B is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 3A following rotation of the plunger.

FIG. 3C is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 3B following depression of the plunger.

FIG. 4A is a still further exemplary partial cross-sectional schematicof an assay device.

FIG. 4B is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 4A following rotation of the plunger.

FIG. 4C is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 4B following depression of the plunger.

FIG. 5A is yet another exemplary partial cross-sectional schematic of anassay device.

FIG. 5B is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 5A following depression of the plunger.

FIG. 6A is still another exemplary partial cross-sectional schematic ofan assay device.

FIG. 6B is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 6A following depression of the plunger.

FIG. 7A is a still further exemplary partial cross-sectional schematicof an assay device.

FIG. 7B is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 7A following an initial depression of the plunger.

FIG. 7C is an exemplary partial cross-sectional schematic of the assaydevice of FIG. 7B following a further depression of the plunger.

While the above-identified drawings, which may not be drawn to scale,set forth various embodiments of the present disclosure, otherembodiments are also contemplated, as noted in the Detailed Description.

DETAILED DESCRIPTION

Assay devices and methods of detecting a target analyte are provided.

The recitation of any numerical range by endpoints is meant to includethe endpoints of the range, all numbers within the range, and anynarrower range within the stated range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.8, 4, and 5). Unless otherwise indicated, all numbersexpressing quantities or ingredients, measurement of properties and soforth used in the specification and embodiments are to be understood asbeing modified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in theforegoing specification and attached listing of embodiments can varydepending upon the desired properties sought to be obtained by thoseskilled in the art utilizing the teachings of the present disclosure. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claimed embodiments, eachnumerical parameter should at least be construed in light of the numberof reported significant digits and by applying ordinary roundingtechniques.

For the following Glossary of defined terms, these definitions shall beapplied for the entire application, unless a different definition isprovided in the claims or elsewhere in the specification.

Glossary

Certain terms are used throughout the description and the claims that,while for the most part are well known, may require some explanation. Itshould be understood that, as used herein:

The term “a”, “an”, and “the” are used interchangeably with “at leastone” to mean one or more of the elements being described.

The term “and/or” means either or both. For example, the expression “Aand/or B” means A, B, or a combination of A and B.

The term “antibody” refers to an immunoglobulin molecule that reactswith a specific antigen, such as a target analyte, and further refers toan antibody fragment (e.g., ScFv or Fab).

The term “antigen” refers to any substance capable of inducing aspecific immune response and of reacting with the products of thatresponse.

The term “aptamer” refers to a DNA or RNA molecule or a peptide thatbinds to a specific target analyte molecule or atom.

The term “ligand” refers to an atom, molecule, functional group, or ionthat is bound to a target analyte molecule or atom, thereby forming acomplex.

The term “affinity component” refers to a material that binds to atarget analyte, for instance an antibody, aptamer, or ligand for thetarget analyte. The affinity component is made up of a binding portionand a detection portion. The binding portion includes an antibody, anucleotide aptamer, a peptide aptamer, or a ligand. The detectionportion includes an enzyme, a catalyst, and/or a molecule that providesan optical signal (e.g., a colorimetric signal, a fluorescent signal, ora luminescent signal).

The term “capture compound” refers to a material that binds to a targetanalyte, for instance an antibody, aptamer, or ligand for the targetanalyte, and is immobilized to bind the target analyte in place.

Analytes of interest, for example bacteria, proteins, and other atoms ormolecules, are often not inherently chemiluminescent, fluorescent,colored, etc., thus in order to achieve specific detection it isnecessary to incorporate an affinity component into the assay device,such as an antibody, ligand or aptamer, that will allow the isolation ofthe analyte and its subsequent detection by a reaction.

In a first aspect, an assay device is provided, comprising:

-   -   a receptacle including a sample entry port;    -   a plunger disposed within the receptacle and having a first        surface;    -   at least one reagent;    -   a membrane attached to the first surface of the plunger, the        membrane comprising:        -   a target conjugate zone in which a plurality of affinity            components are disposed;        -   a wick providing capillary force; and        -   a detection zone in which a plurality of capture compounds            are immobilized, wherein the detection zone is located            between the target conjugate zone and the wick.

Referring to FIG. 1A, in an exemplary embodiment an assay device 100comprises a receptacle 12 comprising a sample entry port 14; a plunger16 disposed within the receptacle 12 and having a first surface 17; atleast one reagent 18; a membrane 20 attached to the first surface 17 ofthe plunger 16, the membrane comprising a target conjugate zone 22 inwhich a plurality of affinity components 23 are disposed; a wick 24providing capillary force; and a detection zone 26 in which a pluralityof capture compounds 27 are immobilized. The detection zone 26 islocated between the target conjugate zone 22 and the wick 24. In certainembodiments, the at least one reagent 18 is disposed in the receptacle12 and the receptacle 12 further comprises a barrier 28 to separate theat least one reagent 18 from the plunger 16. Optionally, the barriercomprises a metal foil or a pouch, for instance a pouch inside of whichat least one reagent is disposed. In certain embodiments, the plunger issecured in the receptacle using a compression fitting, glue, or a ridgeformed on the interior of the receptacle wall.

Referring to FIG. 1B, when a force F is applied to the plunger 16, theplunger 16 is configured to detach the detection zone 26 from being influid communication with the wick 24 and place the detection zone 26 influid communication with the at least one reagent 18. In certainembodiments, the plunger 16 comprises an end 30 configured to pierce thebarrier 28, such as a pointed end. The configuration of the end of theplunger can vary depending on the thickness and type of material of thebarrier. In the embodiment illustrated in FIG. 1B, the barrier 28 is afoil pouch and the end 30 of the plunger 16 is rounded.

In a second aspect, an assay device is provided, comprising:

-   -   a receptacle comprising a surface and a sample entry port;    -   a membrane attached to the surface of the receptacle, the        membrane comprising a wick providing capillary force;    -   at least one reagent disposed in the receptacle;    -   a plunger disposed within the receptacle and having a surface;        and    -   a detection zone in which a plurality of capture compounds are        immobilized, the detection zone attached to the surface of the        plunger; wherein the detection zone is located between the        sample entry port and the wick.

Referring to FIGS. 5A and 5B, an assay device 500 is illustrated. Theassay device 500 comprises a receptacle 12 comprising a surface 10 and asample entry port 14; a membrane 20 attached to the surface 10 of thereceptacle 12, the membrane 20 comprising a wick 24 providing capillaryforce; at least one reagent 18 disposed in the receptacle 12; a plunger16 disposed within the receptacle 12 and having a surface 17; and adetection zone 26 in which a plurality of capture compounds 27 areimmobilized, the detection zone 26 attached to the surface 17 of theplunger 16; wherein the detection zone 26 is located between the sampleentry port 14 and the wick 24. When a force F is applied to the plunger16, the detection zone 26 is moved from being in fluid communicationwith the membrane 20 to being in fluid communication with the at leastone reagent 18.

An advantage to the assay devices 100 and 500 illustrated in FIGS. 1A-1Band 5A-5B, respectively, is that a single and simple force F (i.e.,depression of the plunger 16), is capable of moving the detection zone26 from being in fluid communication with the membrane 20 to being influid communication with the at least one reagent 18. Moreover, in theassay device 500 of FIGS. 5A-5B, there is no direct path for the atleast one reagent 18 to be transported up to the wick 24, and thusbackground signal from the at least one reagent 18 reacting with excessaffinity compound will be avoided.

The below disclosure relates to both the first aspect and the secondaspect provided above.

The affinity components employed in the assay devices disclosed hereinare not particularly limited as long as they will bind to the targetanalyte(s) of interest and are capable of participating in a detectionreaction. The affinity components comprise any combination of a bindingportion with a detection portion. As noted above, the binding portionincludes an antibody, a nucleotide aptamer, a peptide aptamer, or aligand, and the detection portion includes an enzyme, a catalyst, and/ora molecule that provides an optical signal (e.g., a colorimetric signal,a fluorescent signal, or a luminescent signal). Some suitable detectionportions of affinity components include for example and withoutlimitation, horseradish peroxidase, luciferase, and alkalinephosphatase. Similar to the affinity components, the capture compoundsare not limited as long as they will both remain fixed in place in thedetection zone and bind to the target analyte(s) of interest. Thecapture compounds typically comprise an aptamer, a peptide, an antibody,a ligand, or a combination thereof.

To generate a detectable signal (e.g., light from a chemiluminescent orfluorescent reaction, light absorbance from colorimetry, etc.), anycaptured analyte(s) and their bound affinity components are reacted withat least one reagent. In many embodiments, the at least one reagentcomprises hydrogen peroxide and/or luciferin. Preferably, the at leastone reagent comprises an aqueous solution. Luminol is often employedwith chemiluminescent reactions involving hydrogen peroxide, thus incertain aspects of the disclosure, the barrier comprises a pouch and thepouch contains luminol. One suitable pouch comprises a cup structurehaving two plastic rings with a layer of foil affixed to and coveringeach ring, to form an enclosed cup. The pouch prevents the luminol andhydrogen peroxide from reacting prior to the addition of the capturedtarget analyte, but once the pouch is pierced, the luminol is allowed tocontact the hydrogen peroxide. In certain embodiments, the affinitycomponents comprise an enzyme conjugated to an aptamer or an enzymeconjugated to an antibody. The skilled practitioner can identifyaptamers and antibodies for specific target analytes of interest. Commonenzymes include for example horseradish peroxidase and luciferase. Inaspects including the horseradish peroxidase enzyme conjugated to anaptamer or antibody, the quantitative determination of a target analytewill result from the amount of conjugated horseradish peroxidase thatreacts with luminol and hydrogen peroxide, for instance. An advantage ofthe use of the membrane in the receptacle, is that it minimizesbackground signal by removing excess reaction catalyst (e.g.,horseradish peroxidase) via continued travel of the catalyst in solutionby capillary action towards the wick and away from the at least onereagent.

The membrane optionally comprises one or more materials typicallyemployed in flow assays, for example and without limitation, a fluidcontrol film, a capillary fluid conductor (e.g., a stack of fluidcontrol films), nitrocellulose, or other nonwoven membranes, such asmembranes made of nylon, polysulfone, polyethersulfone, orpolyvinylidenedifluoride (PVDF). Suitable nitrocellulose and PVDFmembranes, for example, are known to the skilled practitioner, and arecommercially available from suppliers such as Bio-Rad Laboratories, Inc.(Hercules, Calif.). The detection zone of a membrane optionallycomprises a nonwoven membrane or similar material having a tortuous flowpath to maximize contact between the target analyte in a sample solutionand the capture compound.

A fluid control film comprises a sheet having at least onemicrostructure-bearing surface with one or more channels therein thatpermits, promotes, or facilitates control or directional flow of aliquid. Suitable fluid control films are described in U.S. Pat. Nos.5,514,120; 5,728,446; 6,080,243; and 6,290,685. Fluid control films aretypically in the form of sheets or films rather than a mass of fibers.The channels of fluid control films provide more effective liquid flowthan is generally achieved with webs, foam, or tows formed from fibers.The walls of channels formed in fibers will exhibit relatively randomundulations and complex surfaces that interfere with flow of liquidthrough the channels. In contrast, the channels are precisely replicatedfrom a predetermined pattern and form a series of individual opencapillary channels that extend along a major surface. Thesemicroreplicated channels formed in sheets, films, or tubes arepreferably uniform and regular along substantially each channel lengthand more preferably from channel to channel. Fluid typically wicks upfluid control films a distance of about 2.5 inches (6.35 cm) withinapproximately 15 seconds. Advantages of employing a fluid control filmas compared to a nonwoven membrane include a more even flow path, andthus more reproducible results.

Fluid control films can be formed from any thermoplastic materialssuitable for casting, or embossing including, for example, polyolefins,polyesters, polyamides, poly(vinyl chloride), polyether esters,polyimides, polyesteramide, polyacrylates, polyvinylacetate, hydrolyzedderivatives of polyvinylacetate, etc., or combinations thereof.Polyolefins are commonly employed, particularly polyethylene orpolypropylene, blends and/or copolymers thereof, and copolymers ofpropylene and/or ethylene with minor proportions of other monomers, suchas vinyl acetate or acrylates such as methyl and butylacrylate.Polyolefins exhibit excellent physical properties, ease of processing,and typically lower cost than other thermoplastic materials havingsimilar characteristics. Polyolefins readily replicate the surface of acasting or embossing roll. They are tough, durable and hold their shapewell, thus making such films easy to handle after the casting orembossing process. Hydrophilic polyurethanes also have advantageousphysical properties and inherently high surface energy. Alternatively,fluid control films can be cast from thermosets (curable resinmaterials) such as polyurethanes, acrylates, epoxies and silicones, andcured by exposure to heat or UV or E-beam radiation, or moisture. Thesematerials may contain various additives including surface energymodifiers (such as surfactants and hydrophilic polymers), plasticizers,antioxidants, pigments, release agents, antistatic agents and the like.Suitable fluid control films also can be manufactured using pressuresensitive adhesive materials. In some cases the channels may be formedusing inorganic materials (e.g., glass, ceramics, or metals).Preferably, the fluid control film substantially retains its geometryand surface characteristics upon exposure to liquids.

In many embodiments, the plunger of the assay device further comprises asecond surface opposite the first surface and an end in communicationwith the first surface and the second surface, wherein the membrane isin contact with the end and with the second surface. For instance, FIG.1A illustrates a second surface 29 of the plunger 16 opposite the firstsurface 17 of the plunger 16 and an end 30 in communication with thefirst surface 17 and the second surface 29, wherein the membrane 20 isin contact with the end 30 and with the second surface 29. In theembodiment shown in FIG. 1A, the membrane 20 comprises a U-shape aroundthe plunger 16. Other shapes of the membrane are also contemplated, suchas a V-shape, a squared-off U shape, and the like. Often, the detectionzone of the membrane is located at the end of the plunger. Referring toeach of FIGS. 1A, 2A, 3A, 5A, and 6A, having a detection zone 26 locatedat the end 30 of the plunger 16 places the detection zone in relativelyclose proximity to the at least one reagent. In certain aspects, thereceptacle 12 comprises a test tube or a shape similar to a test tube.When the assay device is placed in a meter such as a 3M CLEAN-TRACE NGLuminometer (available from 3M Company, St. Paul, Minn.), the end 30 ofthe plunger 16 will be in proximity to the detection zone of theluminometer.

In certain embodiments, the assay device further includes a component(not shown) for assisting in piercing a barrier, for instance a shuttleas described in published international patent application WO 97/23596.The component includes one or more of: a pointed end, at least oneprojection on the end, and at least one aperture in a surface of thecomponent. Typically, such a component would be advantageously employedwhen the end of the plunger is rounded or squared-off or the detectionzone is located at the end of the plunger. An exemplary configuration ofan assay device having such a component includes the component disposedbetween the bottom of the plunger and the barrier above the reagentsolution. The component may be separately movable to break the barrier,or may be attached to the plunger so that when the plunger ismanipulated the component pierces the barrier in advance of the plungerreaching the barrier. In certain embodiments, the component is coatedwith, or otherwise contains, a solid reagent to be added to the reactionmixture when the component breaks the barrier and comes into contactwith the reagent solution.

Optionally, the membrane further comprises a control line disposedbetween the wick and the detection zone, the control line comprising ageneric capture compound. Referring to FIG. 3A, the assay device 300 isillustrated to include a control line 32 on the membrane 20 between thewick 24 and the detection zone 26. The generic capture compoundtypically provides a color change when excess affinity component reactswith the generic capture compound. The control line may be easily viewedthrough a window 34, which allows a user to determine visibly if thesample has traveled past the detection zone and any captured targetanalyte is ready to be detected. Alternatively, the wick 24 (all orpart) could be treated with a weakly buffered solution of a pHindicator. For example, bromophenol blue undergoes a yellow to bluetransition as the pH increases from 3 to 5. For any samples that willhave a pH of 5 or above once buffered, the appearance of a blue colorwill function as a sign the sample is ready for detection of anycaptured target analyte.

FIGS. 3A-3C and 2A-2C further illustrate an assay device 300 and anassay device 200, respectively, including a plunger 16 that contains athreaded lower section 36. The threaded lower section 36 is mounted toan aligner 38 connected to a wall 13 of the receptacle 12 so that whenthe plunger 16 is rotated about its vertical axis a lower portion 40 ofthe membrane 20 does not rotate and the resultant tearing force breaksthe lower portion 40 apart from the rest of the membrane 20. The aligner38 is connected to the wall 13 in such a manner that it can movevertically, for instance with a protrusion in a slot in the wall (notshown), enabling the lower portion 40 to move downwards with therotation of the threaded lower section 36 of the plunger 16, creating aphysical gap in the membrane 20. When the plunger 16 is subsequentlydepressed, the lower portion 40 is able to break the barrier 28 andinteract with the at least one reagent 18; however, the break in themembrane 20 prevents any reaction fluid from travelling up to the wick24 where any excess affinity component 23 has been retained.

Referring to FIGS. 4A-4C, an assay device 400 is illustrated. In thisaspect, the assay device 400 includes a receptacle 12 containing aplunger 16 and a plunger sleeve 19 disposed within the receptacle 12.The device comprises a membrane 20 attached to an exterior surface 15 ofthe plunger sleeve 19. The plunger sleeve 19 is configured to define anopening 42 in a portion of the plunger sleeve 19 adjacent to thedetection zone 26 of the membrane 20. The plunger sleeve 19 is securedin the receptacle 12 in this embodiment by being mounted to an aligner38 connected to a wall 13 of the receptacle 12. The aligner 38 isconnected to the wall 13 in such a manner that it can move vertically,such as being in communication with a vertical slot in the wall (notshown). The detection zone 26 is attached to a surface of the plunger16. A plurality of capture compounds 27 are immobilized on the detectionzone 26. As shown in FIG. 4B, when the plunger 16 is rotated, only thedetection zone 26 of the membrane 20 rotates and the resultant tearingforce breaks the detection zone 26 apart from the rest of the membrane20 and moves the detection zone 26 into the interior of the plungersleeve 19. As shown in FIG. 4C, when the plunger 16 is subsequentlydepressed, the end of the plunger 30 pierces the barrier 28 and thedetection zone 26 is moved through the broken barrier 28 and into fluidcommunication with the at least one reagent 18 contained within thereceptacle 12.

Additionally, the plunger 16 may also be used to pierce a barrier inwhich the barrier comprises a pouch, and thus enable the simultaneousaddition of extra components to the at least one reagent 18. Themembrane 20 is shown arranged with the wick 24 at the bottom of themembrane 20 and a sample entry port (not shown) at the top. In thisarrangement the sample fluid flows downwards. In a variation of thisaspect, the assay device 400 can be simply modified so that the sampleentry port is placed where the wick 24 is shown in FIGS. 4A-4B with thewick 24 to be relocated to a position above the detection zone 26. Sucha device functions as a dipstick membrane, a common method in which thelower end of the membrane is immersed in the test solution. Potentialsample overfilling is prevented because any excess sample added wouldflow back out of the port.

Referring to each of FIGS. 2B, 3B, and 4B, in certain embodiments of theassay device, when the plunger 16 is rotated within the receptacle 12,the plunger 16 is configured to detach the detection zone 26 from beingin fluid communication with the wick 24.

Referring to FIGS. 6A and 6B, an assay device 600 is provided. Theplunger 16 comprises a spring 44, wherein when the plunger 16 isdepressed, the spring 44 engages with the membrane 20 at the detectionzone 26 and the spring 44 expands to detach the detection zone 26 (onwhich a plurality of capture compounds 27 are immobilized) from being influid communication with the wick 24 and to place the detection zone 26in fluid communication with the at least one reagent 18.

Referring to FIGS. 7A and 7B, an assay device 700 is provided. Theplunger 16 comprises a spring 44, wherein when the plunger 16 isdepressed, the spring 44 engages with the membrane 20 and the spring 44expands to form a break in the membrane 20 at portion 21 to prevent thedetection zone 26 from continuing to be in fluid communication with thewick 24. Referring to FIG. 7C, when the plunger 16 is further depressed,the detection zone 26 (on which a plurality of capture compounds 27 areimmobilized) is placed in fluid communication with the at least onereagent 18. The break in the membrane 20 at portion 21 minimizes fluidflow of the at least one reagent towards the wick 24, to therebyminimize background signal during detection.

In another embodiment of an assay device (not shown), the at least onereagent is disposed inside the plunger, and the plunger is configured torelease a portion of the at least one reagent to place the portion ofthe at least one reagent in fluid communication with the detection zone.Such a configuration avoids the use of a separate barrier to isolate theat least one reagent from the membrane.

In a further embodiment of an assay device (not shown), a portion of thereceptacle is opaque and a portion of the receptacle is transparent tolight. An advantage to having a portion of the receptacle be opaque isthat detection of background signal beyond the portion of the receptacletransparent to light is minimized.

In a third aspect, a method for detection of a target analyte isprovided, the method comprising:

-   -   (a) providing an assay device comprising:        -   a receptacle comprising a surface and a sample entry port;        -   at least one reagent;        -   a plunger disposed within the receptacle and having a            surface;        -   a membrane attached to the surface of the plunger, the            surface of the receptacle, or both, the membrane comprising            a wick providing capillary force; and        -   a detection zone in which a plurality of capture compounds            are immobilized, wherein the detection zone is located            between the sample entry port and the wick;    -   (b) providing a sample suspected to contain a target analyte;    -   (c) adding the sample onto the membrane through the sample entry        port;    -   (d) allowing the sample to travel along the membrane until the        sample reaches the detection zone;    -   (e) immobilizing the target analyte through reaction of the        target analyte with the capture compounds;    -   (f) reacting the immobilized target analyte with the at least        one reagent to generate a detectable signal; and    -   (g) detecting the generated signal.

Any of the assay devices described in detail above with respect to thefirst and second aspects are suitable for use in the method of the thirdaspect.

In certain embodiments, the providing of the sample further comprisesadding a plurality of affinity components to the sample to conjugate anytarget analyte, whereas in other embodiments, the membrane furthercomprises a target conjugate zone in which a plurality of affinitycomponents are disposed, wherein the target conjugate zone is locatedbetween the sample entry port and the detection zone.

In most embodiments, the detection zone is attached to the surface ofthe plunger and/or attached to the surface of the receptacle. To reactthe immobilized target analyte with the at least one reagent to generatea detectable signal, the reacting further comprises applying force tothe plunger to detach the detection zone from being in fluidcommunication with the wick and place the detection zone in fluidcommunication with the at least one reagent. For instance, applyingforce to the plunger typically comprises rotating the plunger,depressing the plunger, or rotating the plunger followed by depressingthe plunger.

An advantage of the method is that it is compatible with a variety ofquantitative detection methods. For example, in an embodiment thegenerated signal comprises luminescence and the detecting comprisesusing a luminometer to measure the luminescence. In another embodiment,the generated signal comprises fluorescence and the detecting comprisesusing a fluorimeter, and in a further embodiment the generated signalcomprises a color change and the detecting comprises using acolorimeter. Preferably, the detecting further comprises quantifying theluminescence, fluorescence, or color change.

Various items are described that are assay devices or methods ofdetecting a target analyte using the assay devices.

Embodiment 1 is an assay device comprising a receptacle comprising asample entry port; a plunger disposed within the receptacle and having afirst surface; at least one reagent; a membrane attached to the firstsurface of the plunger, the membrane comprising a target conjugate zonein which a plurality of affinity components are disposed; a wickproviding capillary force; and a detection zone in which a plurality ofcapture compounds are immobilized. The detection zone is located betweenthe target conjugate zone and the wick.

Embodiment 2 is the assay device of embodiment 1, wherein when force isapplied to the plunger, the plunger is configured to detach thedetection zone from being in fluid communication with the wick and placethe detection zone in fluid communication with the at least one reagent.

Embodiment 3 is the assay device of embodiment 1 or embodiment 2,wherein the at least one reagent is disposed in the receptacle.

Embodiment 4 is the assay device of any of embodiments 1 to 3, whereinthe receptacle further comprises a barrier to separate the at least onereagent from the plunger.

Embodiment 5 is the assay device of embodiment 4, wherein the barriercomprises a metal foil or a pouch.

Embodiment 6 is the assay device of embodiment 4, wherein the plungerfurther comprises an end configured to pierce the barrier.

Embodiment 7 is the assay device of embodiment 4 or embodiment 5,wherein the plunger comprises a pointed end.

Embodiment 8 is the assay device of embodiment 1, wherein the at leastone reagent is disposed inside the plunger.

Embodiment 9 is the assay device of embodiment 8, wherein the plunger isconfigured to release a portion of the at least one reagent to place theportion of the at least one reagent in fluid communication with thedetection zone.

Embodiment 10 is the assay device of any of embodiments 1 to 9, whereina portion of the receptacle is opaque and a portion of the receptacle istransparent to light.

Embodiment 11 is the assay device of any of embodiments 1 to 10, whereinthe affinity components each comprise a binding portion comprising anantibody, a ligand, a peptide aptamer, a nucleotide aptamer, or acombination thereof.

Embodiment 12 is the assay device of any of embodiments 1 to 11, whereinthe affinity components each comprise a detection portion comprisinghorseradish peroxidase, luciferase, alkaline phosphatase, or acombination thereof.

Embodiment 13 is the assay device of any of embodiments 1 to 12, whereinthe capture compounds comprise an aptamer, a peptide, an antibody, aligand, or a combination thereof.

Embodiment 14 is the assay device of any of embodiments 1 to 13, whereinthe at least one reagent comprises hydrogen peroxide.

Embodiment 15 is the assay device of any of embodiments 1 to 14, whereinthe at least one reagent comprises luciferin.

Embodiment 16 is the assay device of embodiment 15, wherein the barriercomprises a pouch and the pouch contains luminol.

Embodiment 17 is the assay device of any of embodiments 1 to 16, whereinthe affinity components comprise an enzyme conjugated to a peptideaptamer or a nucleotide aptamer.

Embodiment 18 is the assay device of any of embodiments 1 to 16, whereinthe affinity components comprise an enzyme conjugated to an antibody.

Embodiment 19 is the assay device of any of embodiments 1 to 18, whereinthe at least one reagent comprises an aqueous solution.

Embodiment 20 is the assay device of any of embodiments 1 to 19, whereinthe membrane is a fluid control film.

Embodiment 21 is the assay device of any of embodiments 1 to 20, whereinthe membrane comprises nitrocellulose.

Embodiment 22 is the assay device of any of embodiments 1 to 21, whereinthe detection zone comprises a nonwoven membrane.

Embodiment 23 is the assay device of any of embodiments 1 to 7 or 10 to22, wherein when the plunger is rotated within the receptacle, theplunger is configured to detach the detection zone from being in fluidcommunication with the wick.

Embodiment 24 is the assay device of any of embodiments 1 to 18, whereinthe plunger further comprises a second surface opposite the firstsurface and an end in communication with the first surface and thesecond surface, wherein the membrane is in contact with the end and withthe second surface.

Embodiment 25 is the assay device of embodiment 24, wherein thedetection zone is located at the end of the plunger.

Embodiment 26 is the assay device of any of embodiments 1 to 25, whereinthe membrane further comprises a control line disposed between the wickand the detection zone, the control line comprising a generic capturecompound.

Embodiment 27 is the assay device of any of embodiments 1 to 26, whereinthe receptacle comprises a test tube.

Embodiment 28 is the assay device of any of embodiments 1 to 27, whereinthe plunger comprises a spring, wherein when the plunger is depressed,the spring engages with the membrane at the detection zone and thespring expands to detach the detection zone from being in fluidcommunication with the wick and to place the detection zone in fluidcommunication with the at least one reagent.

Embodiment 29 is the assay device of any of embodiments 1 to 27, whereinthe membrane comprises a capillary fluid conductor.

Embodiment 30 is the assay device of embodiment 29, wherein thecapillary fluid conductor comprises a stacked fluid transport film.

Embodiment 31 is an assay device comprising a receptacle comprising asurface and a sample entry port; a membrane attached to the surface ofthe receptacle, the membrane comprising a wick providing capillaryforce; at least one reagent disposed in the receptacle; a plungerdisposed within the receptacle and having a surface; and a detectionzone in which a plurality of capture compounds are immobilized, thedetection zone attached to the surface of the plunger; wherein thedetection zone is located between the sample entry port and the wick.

Embodiment 32 is the assay device of embodiment 31, wherein when a forceis applied to the plunger, the detection zone is moved from being influid communication with the membrane to being in fluid communicationwith the reagent.

Embodiment 33 is the assay device of embodiment 31 or embodiment 32,wherein a portion of the receptacle is opaque and a portion of thereceptacle is transparent to light.

Embodiment 34 is the assay device of any of embodiments 31 to 33,wherein the affinity components each comprise a binding portioncomprising an antibody, a ligand, a peptide aptamer, a nucleotideaptamer, or a combination thereof.

Embodiment 35 is the assay device of any of embodiments 31 to 34,wherein the affinity components each comprise a detection portioncomprising horseradish peroxidase, luciferase, alkaline phosphatase, ora combination thereof.

Embodiment 36 is the assay device of any of embodiments 31 to 35,wherein the capture compounds comprise an aptamer, a peptide, anantibody, a ligand, or a combination thereof.

Embodiment 37 is the assay device of any of embodiments 31 to 36,wherein the at least one reagent comprises hydrogen peroxide.

Embodiment 38 is the assay device of any of embodiments 31 to 37,wherein the at least one reagent comprises luciferin.

Embodiment 39 is the assay device of any of embodiments 31 to 38,wherein the affinity components comprise an enzyme conjugated to apeptide aptamer or a nucleotide aptamer.

Embodiment 40 is the assay device of any of embodiments 31 to 38,wherein the affinity components comprise an enzyme conjugated to anantibody.

Embodiment 41 is the assay device of any of embodiments 31 to 40,wherein the at least one reagent comprises an aqueous solution.

Embodiment 42 is the assay device of any of embodiments 31 to 41,wherein the membrane is a fluid control film.

Embodiment 43 is the assay device of any of embodiments 31 to 42,wherein the membrane comprises nitrocellulose.

Embodiment 44 is the assay device of any of embodiments 31 to 43,wherein the detection zone comprises a nonwoven membrane.

Embodiment 45 is the assay device of any of embodiments 31 to 44,wherein when the plunger is rotated within the receptacle, the plungeris configured to detach the detection zone from being in fluidcommunication with the wick and place the detection zone in fluidcommunication with the at least one reagent.

Embodiment 46 is the assay device of any of embodiments 31 to 45,wherein the receptacle further comprises a barrier to separate the atleast one reagent from the plunger.

Embodiment 47 is the assay device of embodiment 46, wherein the barriercomprises a metal foil or a pouch.

Embodiment 48 is the assay device of embodiment 47, wherein the plungerfurther comprises an end configured to pierce the barrier.

Embodiment 49 is the assay device of embodiment 47 or embodiment 48,wherein the plunger comprises a pointed end.

Embodiment 50 is the assay device of embodiment 38, wherein the barriercomprises a pouch and the pouch contains luminol.

Embodiment 51 is the assay device of any of embodiments 31 to 50,wherein the membrane further comprises a control line disposed betweenthe wick and the detection zone, the control line comprising a genericcapture compound.

Embodiment 52 is the assay device of any of embodiments 31 to 51,wherein the receptacle comprises a test tube.

Embodiment 53 is a method for detection of a target analyte, the methodcomprising (a) providing an assay device comprising a receptaclecomprising a surface and a sample entry port; at least one reagent; aplunger disposed within the receptacle and having a surface; a membraneattached to the surface of the plunger, the surface of the receptacle,or both, the membrane comprising a wick providing capillary force; and adetection zone in which a plurality of capture compounds areimmobilized, wherein the detection zone is located between the sampleentry port and the wick; (b) providing a sample suspected to contain atarget analyte; (c) adding the sample onto the membrane through thesample entry port; (d) allowing the sample to travel along the membraneuntil the sample reaches the detection zone; (e) immobilizing the targetanalyte through reaction of the target analyte with the capturecompounds; (f) reacting the immobilized target analyte with the at leastone reagent to generate a detectable signal; and (g) detecting thegenerated signal.

Embodiment 54 is the method of embodiment 53, wherein the providing thesample further comprises adding a plurality of affinity components tothe sample to conjugate any target analyte.

Embodiment 55 is the method of embodiment 53, wherein the membranefurther comprises a target conjugate zone in which a plurality ofaffinity components are disposed, the target conjugate zone locatedbetween the sample entry port and the detection zone.

Embodiment 56 is the method of any of embodiments 53 to 55, wherein thereacting further comprises applying force to the plunger to detach thedetection zone from being in fluid communication with the wick and placethe detection zone in fluid communication with the at least one reagent.

Embodiment 57 is the method of embodiment 56, wherein applying force tothe plunger comprises rotating the plunger.

Embodiment 58 is the method of embodiment 56, wherein applying force tothe plunger comprises depressing the plunger.

Embodiment 59 is the method of any of embodiments 53 to 58, wherein thedetection zone is attached to the surface of the plunger.

Embodiment 60 is the method of any of embodiments 53 to 58, wherein thedetection zone is attached to the surface of the receptacle.

Embodiment 61 is the method of any of embodiments 53 to 60, wherein thegenerated signal comprises luminescence and wherein the detectingcomprises using a luminometer to measure the luminescence.

Embodiment 62 is the method of embodiment 61, wherein the detectingfurther comprises quantifying the luminescence.

Embodiment 63 is the method of any of embodiments 53 to 60, wherein thegenerated signal comprises fluorescence.

Embodiment 64 is the method of embodiment 63, wherein the detectingfurther comprises quantifying the fluorescence.

Embodiment 65 is the method of any of embodiments 53 to 60, wherein thegenerated signal comprises a color change.

Embodiment 66 is the method of embodiment 65, wherein the detectingfurther comprises quantifying the color change.

Embodiment 67 is the method of any of embodiments 53 to 66, wherein aportion of the receptacle is opaque and a portion of the receptacle istransparent to light.

Embodiment 68 is the method of any of embodiments 53 to 67, wherein theaffinity components each comprise a binding portion comprising anantibody, a ligand, a peptide aptamer, a nucleotide aptamer, or acombination thereof.

Embodiment 69 is the method of any of embodiments 53 to 68, wherein theaffinity components each comprise a detection portion comprisinghorseradish peroxidase, luciferase, alkaline phosphatase, or acombination thereof.

Embodiment 70 is the method of any of embodiments 53 to 69, wherein thecapture compounds comprise an aptamer, a peptide, an antibody, a ligand,or a combination thereof.

Embodiment 71 is the method of any of embodiments 53 to 70, wherein theat least one reagent comprises hydrogen peroxide.

Embodiment 72 is the method of any of embodiments 53 to 71, wherein theat least one reagent comprises luciferin.

Embodiment 73 is the method of any of embodiments 53 to 71, wherein theaffinity components comprise an enzyme conjugated to a peptide aptameror a nucleotide aptamer.

Embodiment 74 is the method of any of embodiments 53 to 71, wherein theaffinity components comprise an enzyme conjugated to an antibody.

Embodiment 75 is the method of any of embodiments 53 to 74, wherein theat least one reagent comprises an aqueous solution.

Embodiment 76 is the method of any of embodiments 53 to 75, wherein themembrane is a fluid control film.

Embodiment 77 is the method of any of embodiments 53 to 76, wherein themembrane comprises nitrocellulose.

Embodiment 78 is the method of any of embodiments 53 to 77, wherein thedetection zone comprises a nonwoven membrane.

Embodiment 79 is the method of any of embodiments 53 to 78, wherein whenthe plunger is rotated within the receptacle, the plunger is configuredto detach the detection zone from being in fluid communication with thewick and place the detection zone in fluid communication with the atleast one reagent.

Embodiment 80 is the method of embodiment 53, wherein the at least onereagent is disposed in the receptacle.

Embodiment 81 is the method of any of embodiments 53 to 80, wherein thereceptacle further comprises a barrier to separate the at least onereagent from the plunger.

Embodiment 82 is the method of embodiment 81, wherein the barriercomprises a metal foil or a pouch.

Embodiment 83 is the method of embodiment 82, wherein the barriercomprises a pouch and the pouch contains luminol.

Embodiment 84 is the method of embodiment 82, wherein the plungerfurther comprises an end configured to pierce the barrier.

Embodiment 85 is the method of embodiment 82 or embodiment 83, whereinthe plunger comprises a pointed end.

Embodiment 86 is the method of embodiment 53, wherein the at least onereagent is disposed inside the plunger.

Embodiment 87 is the method of embodiment 86, wherein the plunger isconfigured to release a portion of the at least one reagent to place theportion of the at least one reagent in fluid communication with thedetection zone.

Embodiment 88 is the method of any of embodiments 53 to 87, wherein theplunger further comprises a second surface opposite the first surfaceand an end in communication with the first surface and the secondsurface, wherein the membrane is in contact with the end and with thesecond surface.

Embodiment 89 is the method of embodiment 88, wherein the detection zoneis located at the end of the plunger.

Embodiment 90 is the method of any of embodiments 53 to 89, wherein themembrane further comprises a control line disposed between the wick andthe detection zone, the control line comprising a generic capturecompound.

Embodiment 91 is the method of any of embodiments 53 to 90, wherein thereceptacle comprises a test tube.

Embodiment 92 is the method of any of embodiments 53 to 91, wherein theplunger comprises a spring, wherein when the plunger is depressed, thespring engages with the membrane at the detection zone and the springexpands to detach the detection zone from being in fluid communicationwith the wick and to place the detection zone in fluid communicationwith the at least one reagent.

Embodiment 93 is the method of any of embodiments 53 to 92, wherein themembrane comprises a capillary fluid conductor.

Embodiment 94 is the method of embodiment 93, wherein the capillaryfluid conductor comprises a stacked fluid transport film.

EXAMPLES

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention. These examplesare for illustrative purposes only and are not meant to be limiting onthe scope of the appended claims.

Materials

Unless otherwise noted, all parts, percentages, ratios, etc., in theexamples and in the remainder of the specification are by weight. Unlessotherwise noted, all chemicals are available from chemical supplierssuch as Sigma-Aldrich Chemical Company, St. Louis, Mo.

Example 1-1

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the plunger. Capture antibodies to PenicillinG are deposited on the detection zone. Luminol is contained within afoil pouch positioned below the plunger and separating the plunger froma reagent solution containing hydrogen peroxide and, optionally, asurfactant. A 100 μL aliquot of the sample solution is introduced via apipet through the sample entry port of the receptacle, and allowed towick along the fluid transport film until it reaches the wick on theother side, typically 5 minutes. During this time, any Penicillin Gpresent in the sample has sufficient time to react with theHRP-conjugated aptamers, travel along the film and then bind to thecapture antibody where it will be retained in the detection zone. After5 minutes, the plunger is depressed, puncturing the foil seal andallowing luminol contained within the pouch to contact the reagentsolution. Any HRP retained in the detection zone facilitates theoxidation of luminol by hydrogen peroxide and generates 3-aminophthalatewith the emission of light. The light is measured by a hand-heldluminometer such as the 3M CLEAN-TRACE luminometer (3M Company, St.Paul, Minn.).

Example 1-2

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the plunger. Capture antibodies to PenicillinG are deposited on the detection zone. ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is containedwithin a foil pouch positioned below the plunger and separating theplunger from a reagent solution containing hydrogen peroxide and,optionally, a surfactant. A 200 μL aliquot of the sample solution isintroduced via a pipet through the sample entry port of the receptacle,and allowed to wick along the fluid transport film until it reaches thewick on the other side, typically 5 minutes. During this time, anyPenicillin G present in the sample has sufficient time to react with theHRP-conjugated aptamers, travel along the film and then bind to thecapture antibody where it will be retained in the detection zone. After5 minutes, the plunger is depressed, puncturing the foil seal andallowing the ABTS contained within the pouch to contact the reagentsolution. HRP retained in the detection zone oxidizes the colorless ABTSto its blue radical cation. The intensity of the blue color can beassessed visually by comparison with a color chart, or with acolorimeter such as the 3M CLEAN-TRACE colorimeter (3M Company, St.Paul, Minn.).

Example 1-3

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the plunger. Capture antibodies to PenicillinG are deposited on the detection zone. ADHP(10-acetyl-3,7-dihydroxyphenoxazine) is contained within a foil pouchpositioned below the plunger and separating the plunger from a reagentsolution containing hydrogen peroxide and, optionally, a surfactant. A250 μL aliquot of the sample solution is introduced via a pipet throughthe sample entry port of the receptacle, and allowed to wick along thefluid transport film until it reaches the wick on the other side,typically 5 minutes. During this time, any Penicillin G present in thesample has sufficient time to react with the HRP-conjugated aptamers,travel along the film and then bind to the capture antibody where itwill be retained in the detection zone. After 5 minutes, the plunger isdepressed, puncturing the foil seal and allowing the ADHP containedwithin the pouch to contact the reagent solution. HRP retained in thedetection zone oxidizes the nonfluorescent ADHP to highly fluorescentresorufin. The intensity of the fluorescence can be assessed visually byirradiation with a standard UV lamp, or preferably with a fluorimeterusing an excitation wavelength at of ˜570 nm and emission of ˜585 nm.

Example 1-4

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the plunger. Capture antibodies to PenicillinG are deposited on the detection zone. Luminol is contained within afoil pouch positioned below the plunger and separating the plunger froma reagent solution containing hydrogen peroxide and, optionally, asurfactant. A 100 μL aliquot of the sample solution is introduced via apipet through the sample entry port of the receptacle, and allowed towick along the fluid transport film until it reaches the wick on theother side, typically 5 minutes. During this time, any Penicillin Gpresent in the sample has sufficient time to react with theHRP-conjugated aptamers, travel along the film and then bind to thecapture antibody where it will be retained in the detection zone. A 1 mLaliquot of pH 7 buffer is then added through the sample entry port inorder to rinse the fluid transport film and detection zone of excessHRP-conjugated antibodies. After 5 minutes, the plunger is depressed,puncturing the foil seal and allowing luminol contained within the pouchto contact the reagent solution. Any HRP retained in the detection zonefacilitates the oxidation of luminol by hydrogen peroxide and generates3-aminophthalate with the emission of light. The light is measured by ahand-held luminometer such as the 3M CLEAN-TRACE luminometer (3MCompany, St. Paul, Minn.).

Example 1-5

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the plunger. Capture antibodies to PenicillinG are deposited on the detection zone. ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is containedwithin a foil pouch positioned below the plunger and separating theplunger from a reagent solution containing hydrogen peroxide and,optionally, a surfactant. A 200 μL aliquot of the sample solution isintroduced via a pipet through the sample entry port of the receptacle,and allowed to wick along the fluid transport film until it reaches thewick on the other side, typically 5 minutes. During this time, anyPenicillin G present in the sample has sufficient time to react with theHRP-conjugated aptamers, travel along the film and then bind to thecapture antibody where it will be retained in the detection zone. A 1 mLaliquot of pH 7 buffer is then added through the sample entry port inorder to rinse the fluid transport film and detection zone of excessHRP-conjugated antibodies. After 5 minutes, the plunger is depressed,puncturing the foil seal and allowing the ABTS contained within thepouch to contact the reagent solution. HRP retained in the detectionzone oxidizes the colorless ABTS to its blue radical cation. Theintensity of the blue color can be assessed visually by comparison witha color chart, or with a colorimeter such as the 3M CLEAN-TRACEcolorimeter (3M Company, St. Paul, Minn.).

Example 1-6

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the plunger. Capture antibodies to PenicillinG are deposited on the detection zone. ADHP(10-acetyl-3,7-dihydroxyphenoxazine) is contained within a foil pouchpositioned below the plunger and separating the plunger from a reagentsolution containing hydrogen peroxide and, optionally, a surfactant. A250 μL aliquot of the sample solution is introduced via a pipet throughthe sample entry port of the receptacle, and allowed to wick along thefluid transport film until it reaches the wick on the other side,typically 5 minutes. During this time, any Penicillin G present in thesample has sufficient time to react with the HRP-conjugated aptamers,travel along the film and then bind to the capture antibody where itwill be retained in the detection zone. A 1 mL aliquot of pH 7 buffer isthen added through the sample entry port in order to rinse the fluidtransport film and detection zone of excess HRP-conjugated antibodies.After 5 minutes, the plunger is depressed, puncturing the foil seal andallowing the ADHP contained within the pouch to contact the reagentsolution. HRP retained in the detection zone oxidizes the nonfluorescentADHP to highly fluorescent resorufin. The intensity of the fluorescencecan be assessed visually by irradiation with a standard UV lamp, orpreferably with a fluorimeter using an excitation wavelength at of ˜570nm and emission of ˜585 nm.

Example 2-1

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. Luminol is contained within a foil pouch positionedbelow the plunger and separating the plunger from a reagent solutioncontaining hydrogen peroxide and, optionally, a surfactant. A 100 μLaliquot of the sample solution is introduced via a pipet through thesample entry port of the receptacle, and allowed to wick along the fluidtransport film until it reaches the wick on the other side. During thistime, any c. diff present in the sample has sufficient time to reactwith the HRP-conjugated antibodies, travel along the film and then bindto the capture antibody where it will be retained in the detection zone.After 5 minutes, the plunger is rotated one full turn to cause a breakin the fluid path of the membrane. The plunger is then depressed,puncturing the foil seal and allowing luminol contained within the pouchto contact the reagent solution. Any HRP retained in the detection zonefacilitates the oxidation of luminol by hydrogen peroxide and generates3-aminophthalate with the emission of light. The light is measured by ahand-held luminometer such as the 3M CLEAN-TRACE luminometer.

Example 2-2

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. Luminol is contained within a foil pouch positionedbelow the plunger and separating the plunger from a reagent solutioncontaining hydrogen peroxide and, optionally, a surfactant. A 100 μLaliquot of the sample solution is introduced via a pipet through thesample entry port of the receptacle, and allowed to wick along the fluidtransport film until it reaches the wick on the other side. During thistime, any c. diff present in the sample has sufficient time to reactwith the HRP-conjugated antibodies, travel along the film and then bindto the capture antibody where it will be retained in the detection zone.A 1 mL aliquot of pH 7 buffer is then added through the sample entryport in order to rinse the fluid transport film and detection zone ofexcess HRP-conjugated antibodies. After 5 minutes, the plunger isrotated one full turn to cause a break in the fluid path of themembrane. The plunger is then depressed, puncturing the foil seal andallowing luminol contained within the pouch to contact the reagentsolution. Any HRP retained in the detection zone facilitates theoxidation of luminol by hydrogen peroxide and generates 3-aminophthalatewith the emission of light. The light is measured by a hand-heldluminometer such as the 3M CLEAN-TRACE luminometer.

Example 2-3

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. Luminol is contained within a foil pouch positionedbelow the plunger and separating the plunger from a reagent solutioncontaining hydrogen peroxide and, optionally, a surfactant. A 100 μLaliquot of the sample solution is introduced via a pipet through thesample entry port of the receptacle, and allowed to wick along the fluidtransport film until it reaches the wick on the other side. During thistime, any c. diff present in the sample has sufficient time to reactwith the HRP-conjugated antibodies, travel along the film and then bindto the capture antibody where it will be retained in the detection zone.A 1 mL aliquot of pH 5 buffer is then added through the sample entryport in order to rinse the fluid transport film and detection zone ofexcess HRP-conjugated antibodies. After 5 minutes, the plunger isrotated one full turn to cause a break in the fluid path of themembrane. The plunger is then depressed, puncturing the foil seal andallowing luminol contained within the pouch to contact the reagentsolution. Any HRP retained in the detection zone facilitates theoxidation of luminol by hydrogen peroxide and generates 3-aminophthalatewith the emission of light. The light is measured by a hand-heldluminometer such as the 3M CLEAN-TRACE luminometer.

Example 2-4

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. Luminol is contained within a foil pouch positionedbelow the plunger and separating the plunger from a reagent solutioncontaining hydrogen peroxide and, optionally, a surfactant. A 100 μLaliquot of the sample solution is introduced via a pipet through thesample entry port of the receptacle, and allowed to wick along the fluidtransport film until it reaches the wick on the other side. During thistime, any c. diff present in the sample has sufficient time to reactwith the HRP-conjugated antibodies, travel along the film and then bindto the capture antibody where it will be retained in the detection zone.A 1 mL aliquot of pH 9 buffer is then added through the sample entryport in order to rinse the fluid transport film and detection zone ofexcess HRP-conjugated antibodies. After 5 minutes, the plunger isrotated one full turn to cause a break in the fluid path of themembrane. The plunger is then depressed, puncturing the foil seal andallowing luminol contained within the pouch to contact the reagentsolution. Any HRP retained in the detection zone facilitates theoxidation of luminol by hydrogen peroxide and generates 3-aminophthalatewith the emission of light. The light is measured by a hand-heldluminometer such as the 3M CLEAN-TRACE luminometer.

Example 2-5

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonicacid]-diammonium salt) is contained within a foil pouch positioned belowthe plunger and separating the plunger from a reagent solutioncontaining hydrogen peroxide and, optionally, a surfactant. A 400 μLaliquot of the sample solution is introduced via a pipet through thesample entry port of the receptacle, and allowed to wick along the fluidtransport film until it reaches the wick on the other side. During thistime, any c. diff present in the sample has sufficient time to reactwith the HRP-conjugated antibodies, travel along the film and then bindto the capture antibody where it will be retained in the detection zone.After 5 minutes, the plunger is rotated one full turn to cause a breakin the fluid path of the membrane. The plunger is then depressed,puncturing the foil seal and allowing ABTS contained within the pouch tocontact the reagent solution. HRP retained in the detection zoneoxidizes the colorless ABTS to its blue radical cation. The intensity ofthe blue color can be assessed visually by comparison with a colorchart, or with a colorimeter such as the 3M CLEAN-TRACE Colorimeter.

Example 2-6

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. ADHP (10-acetyl-3,7-dihydroxyphenoxazine) is containedwithin a foil pouch positioned below the plunger and separating theplunger from a reagent solution containing hydrogen peroxide and,optionally, a surfactant. A 800 μL aliquot of the sample solution isintroduced via a pipet through the sample entry port of the receptacle,and allowed to wick along the fluid transport film until it reaches thewick on the other side. During this time, any c. diff. present in thesample has sufficient time to react with the HRP-conjugated antibodies,travel along the film and then bind to the capture antibody where itwill be retained in the detection zone. After 5 minutes, the plunger isrotated one full turn to cause a break in the fluid path of themembrane. The plunger is then depressed, puncturing the foil seal andallowing ADHP contained within the pouch to contact the reagentsolution. HRP retained in the detection zone oxidizes the nonfluorescentADHP to highly fluorescent resorufin. The intensity of the fluorescencecan be assessed visually by irradiation with a standard UV lamp, orpreferably with a fluorimeter using an excitation wavelength at of ˜570nm and emission of ˜585 nm.

Example 3-1

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. Luminol is contained within a foil pouch positionedbelow the plunger and separating the plunger from a reagent solutioncontaining hydrogen peroxide and, optionally, a surfactant. The wickcontains a small amount of bromophenol blue, in its yellow, acid form. A250 μL aliquot of the sample solution is mixed with 1 mL of pH 7 bufferand introduced via a pipet through the sample entry port of thereceptacle, and allowed to wick along the fluid transport film until itreaches the wick on the other side. This is demonstrated by theappearance of a blue color (due to bromophenol blue) on the wick whichmay be visualized through the window. During this time, any c. diffpresent in the sample has sufficient time to react with theHRP-conjugated antibodies, travel along the film and then bind to thecapture antibody where it will be retained in the detection zone. After5 minutes, the plunger is rotated one full turn to cause a break in thefluid path of the membrane. The plunger is then depressed, puncturingthe foil seal and allowing luminol contained within the pouch to contactthe reagent solution. Any HRP retained in the detection zone facilitatesthe oxidation of luminol by hydrogen peroxide and generates3-aminophthalate with the emission of light. The light is measured by ahand-held luminometer such as the 3M CLEAN-TRACE luminometer.

Example 3-2

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonicacid]-diammonium salt) is contained within a foil pouch positioned belowthe plunger and separating the plunger from a reagent solutioncontaining hydrogen peroxide and, optionally, a surfactant. The wickcontains a small amount of bromophenol blue, in its yellow, acid form. A100 μL aliquot of the sample solution is mixed with 1 mL of pH 7 bufferand introduced via a pipet through the sample entry port of thereceptacle, and allowed to wick along the fluid transport film until itreaches the wick on the other side. This is demonstrated by theappearance of a blue color (due to bromophenol blue) on the wick whichmay be visualized through the window. During this time, any c. diffpresent in the sample has sufficient time to react with theHRP-conjugated antibodies, travel along the film and then bind to thecapture antibody where it will be retained in the detection zone. After5 minutes, the plunger is rotated one full turn to cause a break in thefluid path of the membrane. The plunger is then depressed, puncturingthe foil seal and allowing ABTS contained within the pouch to contactthe reagent solution. HRP retained in the detection zone oxidizes thecolorless ABTS to its blue radical cation. The intensity of the bluecolor can be assessed visually by comparison with a color chart, or witha colorimeter such as the 3M CLEAN-TRACE Colorimeter.

Example 3-3

Antibodies to clostridium difficile as described in U.S. Pat. No.8,697,374 are conjugated with horseradish peroxidase (HRP) and depositedon a nitrocellulose membrane affixed to the surface of the plunger.Capture antibodies to clostridium difficile are deposited on thedetection zone. ADHP (10-acetyl-3,7-dihydroxyphenoxazine) is containedwithin a foil pouch positioned below the plunger and separating theplunger from a reagent solution containing hydrogen peroxide and,optionally, a surfactant. The wick contains a small amount ofbromophenol blue, in its yellow, acid form. A 250 μL aliquot of thesample solution is mixed with 1 mL of pH 7 buffer and introduced via apipet through the sample entry port of the receptacle, and allowed towick along the fluid transport film until it reaches the wick on theother side. This is demonstrated by the appearance of a blue color (dueto bromophenol blue) on the wick which may be visualized through thewindow. During this time, any c. diff present in the sample hassufficient time to react with the HRP-conjugated antibodies, travelalong the film and then bind to the capture antibody where it will beretained in the detection zone. After 5 minutes, the plunger is rotatedone full turn to cause a break in the fluid path of the membrane. Theplunger is then depressed, puncturing the foil seal and allowing ADHPcontained within the pouch to contact the reagent solution. HRP retainedin the detection zone oxidizes the nonfluorescent ADHP to highlyfluorescent resorufin. The intensity of the fluorescence can be assessedvisually by irradiation with a standard UV lamp, or preferably with afluorimeter using an excitation wavelength at of ˜570 nm and emission of˜585 nm.

Example 4-1

Aflatoxin-binding peptides are conjugated with horseradish peroxidase(HRP) and deposited on a nitrocellulose membrane affixed to the surfaceof the plunger. Capture antibodies to aflatoxin are deposited on thedetection zone. The detection zone is bounded on either side by twoweakened regions (for example perforations) which do not inhibit fluidtransport but which will break when physically stressed. Luminol iscontained within a foil pouch positioned below the plunger andseparating the plunger from a reagent solution containing hydrogenperoxide and, optionally, a surfactant. The wick contains a small amountof bromophenol blue, in its yellow, acid form. A 50 μL aliquot of thesample solution is mixed with 0.5 mL of pH 7 buffer and introduced via apipet through the sample entry port of the receptacle, and allowed towick down the fluid transport film until it reaches the wick below. Thisis demonstrated by the appearance of a blue color (due to bromophenolblue) on the wick which may be visualized through the window. Duringthis time, any aflatoxin present in the sample has sufficient time toreact with the HRP-conjugated peptides, travel along the film and thenbind to the capture antibody where it will be retained in the detectionzone. After 5 minutes, the plunger is rotated one quarter turn to causea break in the fluid path of the membrane. The plunger is thendepressed, puncturing the foil seal and allowing luminol containedwithin the pouch to contact the reagent solution and moving thedetection zone of the membrane down into the reagent solution. Any HRPretained in the detection zone facilitates the oxidation of luminol byhydrogen peroxide and generates 3-aminophthalate with the emission oflight. The light is measured by a hand-held luminometer such as the 3MCLEAN-TRACE luminometer.

Example 4-2

Aflatoxin-binding peptides are conjugated with horseradish peroxidase(HRP) and deposited on a nitrocellulose membrane affixed to the surfaceof the plunger. Capture antibodies to aflatoxin are deposited on thedetection zone. The detection zone is bounded on either side by twoweakened regions (for example perforations) which do not inhibit fluidtransport but which will break when physically stressed. Luminol iscontained within a foil pouch positioned below the plunger andseparating the plunger from a reagent solution containing hydrogenperoxide and, optionally, a surfactant. The wick contains a small amountof bromophenol blue, in its yellow, acid form. A 50 μL aliquot of thesample solution is mixed with 0.5 mL of pH 8 buffer and introduced via apipet through the sample entry port of the receptacle, and allowed towick down the fluid transport film until it reaches the wick below. Thisis demonstrated by the appearance of a blue color (due to bromophenolblue) on the wick which may be visualized through the window. Duringthis time, any aflatoxin present in the sample has sufficient time toreact with the HRP-conjugated peptides, travel along the film and thenbind to the capture antibody where it will be retained in the detectionzone. After 5 minutes, the plunger is rotated one quarter turn to causea break in the fluid path of the membrane. The plunger is thendepressed, puncturing the foil seal and allowing luminol containedwithin the pouch to contact the reagent solution and moving thedetection zone of the membrane down into the reagent solution. Any HRPretained in the detection zone facilitates the oxidation of luminol byhydrogen peroxide and generates 3-aminophthalate with the emission oflight. The light is measured by a hand-held luminometer such as the 3MCLEAN-TRACE luminometer.

Example 4-3

Aflatoxin-binding peptides are conjugated with horseradish peroxidase(HRP) and deposited on a nitrocellulose membrane affixed to the surfaceof the plunger. Capture antibodies to aflatoxin are deposited on thedetection zone. The detection zone is bounded on either side by twoweakened regions (for example perforations) which do not inhibit fluidtransport but which will break when physically stressed. Luminol iscontained within a foil pouch positioned below the plunger andseparating the plunger from a reagent solution containing hydrogenperoxide and, optionally, a surfactant. The wick contains a small amountof bromophenol blue, in its yellow, acid form. A 50 μL aliquot of thesample solution is mixed with 0.5 mL of pH 9 buffer and introduced via apipet through the sample entry port of the receptacle, and allowed towick down the fluid transport film until it reaches the wick below. Thisis demonstrated by the appearance of a blue color (due to bromophenolblue) on the wick which may be visualized through the window. Duringthis time, any aflatoxin present in the sample has sufficient time toreact with the HRP-conjugated peptides, travel along the film and thenbind to the capture antibody where it will be retained in the detectionzone. After 5 minutes, the plunger is rotated one quarter turn to causea break in the fluid path of the membrane. The plunger is thendepressed, puncturing the foil seal and allowing luminol containedwithin the pouch to contact the reagent solution and moving thedetection zone of the membrane down into the reagent solution. Any HRPretained in the detection zone facilitates the oxidation of luminol byhydrogen peroxide and generates 3-aminophthalate with the emission oflight. The light is measured by a hand-held luminometer such as the 3MCLEAN-TRACE luminometer.

Example 5-1

Tropomyosin-binding peptides are conjugated with alkaline phosphatase(AP) and deposited on a nitrocellulose membrane affixed to the surfaceof the plunger. Capture antibodies to tropomyosin are deposited on thedetection zone. A chemiluminescent AP substrate such as CDP Star (ABI,Thermo) is contained within a reagent solution optionally containing asurfactant and/or a chemiluminescent enhancer such as Sapphire II orEmerald II. The wick contains a small amount of bromophenol blue, in itsyellow, acid form. A 500 μL aliquot of the sample solution is mixed with0.5 mL of pH 7 buffer and introduced via a pipet through the sampleentry port of the receptacle, and allowed to travel up the fluidtransport film until it reaches the wick at the top. This isdemonstrated by the appearance of a blue color (due to bromophenol blue)on the wick which may be visualized through the window. During thistime, any tropomyosin present in the sample has sufficient time to reactwith the AP-conjugated peptides, travel along the film and then bind tothe capture antibody where it will be retained in the detection zone.After 5 minutes, the plunger is rotated one quarter turn to cause abreak in the fluid path of the membrane. The plunger is then depressed,puncturing the foil seal and moving the detection zone of the membranedown into the reagent solution. Any AP retained in the detection zonehydrolyses the CDP-Star substrate with the emission of light. The lightis measured by a hand-held luminometer such as the 3M CLEAN-TRACEluminometer.

Example 5-2

Tropomyosin-binding peptides are conjugated with alkaline phosphatase(AP) and deposited on a nitrocellulose membrane affixed to the surfaceof the plunger. Capture antibodies to tropomyosin are deposited on thedetection zone. A chemiluminescent AP substrate such as CDP Star (ABI,Thermo) is contained within a reagent solution optionally containing asurfactant and/or a chemiluminescent enhancer such as Sapphire II orEmerald II. The wick contains a small amount of bromophenol blue, in itsyellow, acid form. A 500 μL aliquot of the sample solution is mixed with0.5 mL of pH 7 buffer and introduced via a pipet through the sampleentry port of the receptacle, and allowed to travel up the fluidtransport film until it reaches the wick at the top. This isdemonstrated by the appearance of a blue color (due to bromophenol blue)on the wick which may be visualized through the window. During thistime, any tropomyosin present in the sample has sufficient time to reactwith the AP-conjugated peptides, travel along the film and then bind tothe capture antibody where it will be retained in the detection zone.After 1 minute, the plunger is rotated one quarter turn to cause a breakin the fluid path of the membrane. The plunger is then depressed,puncturing the foil seal and moving the detection zone of the membranedown into the reagent solution. Any AP retained in the detection zonehydrolyses the CDP-Star substrate with the emission of light. The lightis measured by a hand-held luminometer such as the 3M CLEAN-TRACEluminometer.

Example 5-3

Tropomyosin-binding peptides are conjugated with alkaline phosphatase(AP) and deposited on a nitrocellulose membrane affixed to the surfaceof the plunger. Capture antibodies to tropomyosin are deposited on thedetection zone. A chemiluminescent AP substrate such as CDP Star (ABI,Thermo) is contained within a reagent solution optionally containing asurfactant and/or a chemiluminescent enhancer such as Sapphire II orEmerald II. The wick contains a small amount of bromophenol blue, in itsyellow, acid form. A 500 μL aliquot of the sample solution is mixed with0.5 mL of pH 7 buffer and introduced via a pipet through the sampleentry port of the receptacle, and allowed to travel up the fluidtransport film until it reaches the wick at the top. This isdemonstrated by the appearance of a blue color (due to bromophenol blue)on the wick which may be visualized through the window. During thistime, any tropomyosin present in the sample has sufficient time to reactwith the AP-conjugated peptides, travel along the film and then bind tothe capture antibody where it will be retained in the detection zone.After 10 minutes, the plunger is rotated one quarter turn to cause abreak in the fluid path of the membrane. The plunger is then depressed,puncturing the foil seal and moving the detection zone of the membranedown into the reagent solution. Any AP retained in the detection zonehydrolyses the CDP-Star substrate with the emission of light. The lightis measured by a hand-held luminometer such as the 3M CLEAN-TRACEluminometer.

Example 6-1

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the receptacle. Capture antibodies toPenicillin G are deposited on the detection zone. Luminol is containedwithin a foil pouch positioned below the plunger and separating theplunger from a reagent solution containing hydrogen peroxide and,optionally, a surfactant. A 100 μL aliquot of the sample solution isintroduced via a pipet through the sample entry port of the receptacle,and allowed to wick along the fluid transport film until it reaches thewick on the other side, typically 5 minutes. During this time, anyPenicillin G present in the sample has sufficient time to react with theHRP-conjugated aptamers, travel along the film and then bind to thecapture antibody where it will be retained in the detection zone. After5 minutes, the plunger is depressed, puncturing the foil seal andallowing luminol contained within the pouch to contact the reagentsolution. Any HRP retained in the detection zone facilitates theoxidation of luminol by hydrogen peroxide and generates 3-aminophthalatewith the emission of light. The light is measured by a hand-heldluminometer such as the 3M CLEAN-TRACE luminometer.

Example 6-2

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the receptacle. Capture antibodies toPenicillin G are deposited on the detection zone. ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is containedwithin a foil pouch positioned below the plunger and separating theplunger from a reagent solution containing hydrogen peroxide and,optionally, a surfactant. A 100 μL aliquot of the sample solution isintroduced via a pipet through the sample entry port of the receptacle,and allowed to wick along the fluid transport film until it reaches thewick on the other side, typically 5 minutes. During this time, anyPenicillin G present in the sample has sufficient time to react with theHRP-conjugated aptamers, travel along the film and then bind to thecapture antibody where it will be retained in the detection zone. After5 minutes, the plunger is depressed, puncturing the foil seal andallowing the ABTS contained within the pouch to contact the reagentsolution. HRP retained in the detection zone oxidizes the colorless ABTSto its blue radical cation. The intensity of the blue color can beassessed visually by comparison with a color chart, or with acolorimeter such as the 3M CLEAN-TRACE Colorimeter.

Example 6-3

Aptamers that bind specifically to Penicillin G are conjugated withhorseradish peroxidase (HRP) and deposited on a fluid transport filmaffixed to the surface of the receptacle. Capture antibodies toPenicillin G are deposited on the detection zone. ADHP(10-acetyl-3,7-dihydroxyphenoxazine) is contained within a foil pouchpositioned below the plunger and separating the plunger from a reagentsolution containing hydrogen peroxide and, optionally, a surfactant. A100 μL aliquot of the sample solution is introduced via a pipet throughthe sample entry port of the receptacle, and allowed to wick along thefluid transport film until it reaches the wick on the other side,typically 5 minutes. During this time, any Penicillin G present in thesample has sufficient time to react with the HRP-conjugated aptamers,travel along the film and then bind to the capture antibody where itwill be retained in the detection zone. After 5 minutes, the plunger isdepressed, puncturing the foil seal and allowing the ADHP containedwithin the pouch to contact the reagent solution. HRP retained in thedetection zone oxidizes the nonfluorescent ADHP to highly fluorescentresorufin. The intensity of the fluorescence can be assessed visually byirradiation with a standard UV lamp, or preferably with a fluorimeterusing an excitation wavelength at of ˜570 nm and emission of ˜585 nm.

While the specification has described in detail certain exemplaryembodiments, it will be appreciated that those skilled in the art, uponattaining an understanding of the foregoing, may readily conceive ofalterations to, variations of, and equivalents to these embodiments.Furthermore, all publications and patents referenced herein areincorporated by reference in their entirety to the same extent as ifeach individual publication or patent was specifically and individuallyindicated to be incorporated by reference. Various exemplary embodimentshave been described. These and other embodiments are within the scope ofthe following claims.

What is claimed is:
 1. A assay device comprising: a receptaclecomprising a sample entry port; a plunger disposed within the receptacleand having a first surface; at least one reagent; a membrane attached tothe first surface of the plunger, the membrane comprising: a targetconjugate zone in which a plurality of affinity components are disposed;a wick providing capillary force; and a detection zone in which aplurality of capture compounds are immobilized, wherein the detectionzone is located between the target conjugate zone and the wick, whereinthe plunger comprises a spring, wherein when the plunger is depressed,the spring engages with the membrane at the detection zone and thespring expands to detach the detection zone from being in fluidcommunication with the wick and to place the detection zone in fluidcommunication with the at least one reagent.
 2. The assay device ofclaim 1, wherein the at least one reagent is disposed in the receptacle.3. The assay device of claim 1, wherein the receptacle further comprisesa barrier to separate the at least one reagent from the plunger.
 4. Theassay device of claim 1, wherein the affinity components each comprise abinding portion comprising an antibody, a ligand, a peptide aptamer, anucleotide aptamer, or a combination thereof.
 5. The assay device ofclaim 1, wherein the capture compounds comprise an aptamer, a peptide,an antibody, a ligand, or a combination thereof.
 6. The assay device ofclaim 1, wherein the plunger further comprises a second surface oppositethe first surface and an end in communication with the first surface andthe second surface, wherein the membrane is in contact with the end andwith the second surface.
 7. The assay device of claim 1, wherein thereceptacle comprises a test tube.